Research addressing the main specific aims of this project focused on cellular and circuit mechanisms generating the respiratory rhythm and neural activity patterns in the brainstem of rodents. Experimental studies were performed with isolated in situ perfused brainstem-spinal cord and in vitro brainstem slice preparations from neonatal and mature rats. Previously we have identified the brainstem locus (called the pre-Botzinger complex) containing populations of neurons participating in rhythm generation. We have further exploited methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing structural imaging by infrared and differential interference contrast (IR-DIC) microscopy performed simultaneously with functional of activity patterns of the neurons labeled with fluorescence calcium-sensitive dyes and/or fluorescent proteins. This imaging approach has facilitated identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of expression of neuron channels, receptors, and neurotransmitter-related proteins. With these approaches, we have imaged the activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-Botzinger complex and rhythm transmission circuits in vitro, providing the most direct experimental evidence to date that rhythm generation involves an excitatory network of neurons with specialized cellular properties that endow respiratory circuits with multiple mechanisms for producing respiratory oscillations. Methods for imaging by multi-photon laser scanning microscopy that allow three-dimensional reconstruction of the pre-Botzinger complex and other respiratory network components are currently under development. Studies of neuronal synaptic interactions and cellular membrane biophysical properties in the pre-Botzinger complex, including with advanced electrophysiolgical approaches such as the "dynamic clamp", continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratoy rhythm and pattern in the intact mammalian nervous system. These studies have provided additional evidence that neuronal persistent sodium currents and potassium leak conductances represent critical ionic conductance mechanisms for generation and control of respiratory oscillations. Molecular profiling with RT-PCR of messenger RNA expressed in single functionally identified neurons in vitro, as well as immunohistochemical studies, show a profile of sodium, potassium, and neurotransmitter receptor-linked channels consistent with an important role of persistent sodium and potassium leak conductances. We have now identified a specialized class of two-pore domain potassium channels, called TASK channels, that are important contributors to neuronal leak conductance. Electrophysiological studies have also demonstrated that these cellular conductance mechanisms are critically involved in the regulation of rhythmic breathing patterns by a diverse set of endogenous neurochemicals that modulate these conductances as well as by physiological control signals including carbon dioxide and oxygen. A particular focus of these latter studies was elucidating neuromodulatory control of respiratory circuit activity by neurons of the brainstem retrotrapezoid nucleus (RTN), which is critically involved in chemosensory (carbon dioxide-related) regulation, and control by the brainstem serotonergic system, which is postulated to have a critical function in brain state-dependent control of breathing in vivo and is associated with pathophysiological disturbances of breathing such as those underlying sudden infant death syndrome (SIDS). Electrophysiological studies performed in vitro and in situ have established critical functional interactions between raphe and respiratory circuit neurons and have determined the essential modulatory actions of raphe serotonergic neurons in both the neonatal and mature mammalian nervous systems. Raphe neurons were shown to have slow pacemaking properties dependent in part on the kinetic properties of sodium channels, and these pacemaking properties were demonstrated to be essential for continuous modulation of respiratory network excitability and respiratory rhythm generation. In studies employing novel pharmaco-genetic approaches applied in situ and in vivo, RTN neurons that also have slow pacemaking properties were shown to provide a critical excitatory input to core components of the respiratory network for generation and coordination of inspiratory and expiratory neural activity. New models for the operation of brainstem respiratory circuits that incorporate multiple neuromodulatory input control mechanisms have been formulated to explain how specific brainstem circuit components are controlled and regulate patterns of respiratory oscillatory activity. We are currently employing optogenetic approaches for manipulation of activity of specific neuronal populations to further investigate how different populations of network neurons contribute to respiratory pattern generation in various (patho)physiological states.